WO2001055218A1

WO2001055218A1 - Antibodies to the staphylococcal multidrug resistance (smr) protein

Info

Publication number: WO2001055218A1
Application number: PCT/US2001/002996
Authority: WO
Inventors: Rocco Jamie Rotello; Rebecca Ann Farmer
Original assignee: The Procter & Gamble Company
Priority date: 2000-01-31
Filing date: 2001-01-30
Publication date: 2001-08-02
Also published as: AU2001236588A1

Abstract

Described is a novel antibody to the Staphylococcal multidrug resistance (Smr) protein and a hybridoma cell line which produces that antibody. Also described are uses of the antibody, methods for its preparation, compositions comprising the antibody, test kits comprising the antibody, methods for screening for the Smr protein, and a screening method for identifying compounds that bind to the Smr protein.

Description

ANTIBODIES TO THE STAPHYLOCOCCAL MULTIDRUG RESISTANCE (SMR) PROTEIN

CROSS REFERENCE TO RELATED APPLICATION

This application claims the benefit of U S Provisional Application No 60/179,290, filed January 31, 2000

FIELD OF THE INVENTION

This invention relates to novel antibodies, fragments thereof, their uses, and to compositions and products using antibodies More specifically, the invention relates to such antibodies or fragments that selectively bind to Staphylococcal multidrug resistance protein

BACKGROUND

Examples of bacterial infections resistant to antibiotic therapy have been reported in the past, and now are a significant threat to public health in the developed world The development of microbial resistance (perhaps as a result of the intense use of antibacterials over extended periods of time) is of increasing concern in medical science "Resistance" can be defined as existence of organisms, within a population of a given microbial species, that are less susceptible to the action of a given antimicrobial agent This resistance is of particular concern in environments such as hospitals and nursing homes, where relatively high rates of infection and intense use of antibacterials are common See, e g . W Sanders, Jr et al . "Inducible Beta-lactamases Clinical and Epidemiologic Implications for Use of Newer Cephalospoπns", 10 Reviews of Infectious Diseases 830 (1988)

Pathogenic bacteria are known to acquire resistance via several distinct mechanisms including inactivation of the antibiotic by bacterial enzymes (e g , β-lactamases hydrolyzing penicillin and cephalosporms). removal of the antibiotic using efflux pumps, modification of the target of the antibiotic via mutation and genetic recombination (e g , penicillin-resistance in Neiie na gonorrhoeae), and acquisition of a readily transferable gene from an external source to create a resistant target (e g . methicilhn-resistance in Staphylococcus aureus) For example, there are certain Gram positive pathogens, such as vancomycin-resistant Enterococcus faecium. which are resistant to virtually all commercially available antibiotics Bacteria, for instance, demonstrate the surpnsing ability to extrude a variety of lipophihc drugs out of the cytoplasm, S B Levy. 36 Antimicrob Agents Chemother. pp 695-703 (1992) and K Lewis. 19 Trends Biochem Sci. pp 119-123 (1994) In Bacillus subtihs, Staphylococcus aureus and Escherichia cob, genes encoding efflux-mediated multidrug resistance have been identified and sequenced A A Neyfakh, B E Bidnenko and L B Chen 88 Proc Natl Acad Sci U S A , pp 4781-4785 (1992), 0 Lomovskaya and K Lewis 89 Proc Natl Acad Sci U S A , p 8938-8942 (1992), M Midgley 4 Microbiol Sci. pp 125-127 (1987), A S Purewal 68 FEMS Microbiol Lett , pp 73-76 (1991). M Moπmyo, E Hongo. H Hama-Inaba and I Machida 20 Nucleic Acids Res . pp 3159-3165 (1992), B R Lyon and R A Skurray 51 Microbiol Rev , pp 88-134 (1987)) The sequences of these genes suggest that their products should each form 12-14 transmembrane domains See, e g , A A Neyfakh. B E Bidnenko and L B Chen. 88 Proc Natl Acad Sci U S A , pp 4781-4785 (1992), O Lomovskaya and K Lewis, 89 Proc Natl Acad Sci U S A , pp 8938-8942 (1992). D A Rouch, D S Cram. D DiBernardino. G Littlejohn and R A Skurray, 4 Mol Microbiol . pp 2051-2062 (1990), and J M Tennent, B R Lyon. M Midgley, I G Jones, A S Purewal and R A Skurray. 135 J Gen Microbiol . pp 1-10 (1989)

There is also a family of small membrane proteins responsible for the efflux-mediated multidrug resistance in bacteria L Grmius, G Dreguniene, E B Goldberg. D -H Liao and S J Projan, 27 Plasmid. pp 119-129 (1992) These proteins confer resistance to catiomc drugs such as aromatic dyes, e g ethidium bromide, quaternary amines, e g disinfectant benzalkonmm and derivatives of tetraphenylphosphonium See, L Gπnius et al . supra, T G Littlejohn. I T Paulsen, M T Gillespie. J M Tennent. M Midgley, I G Jones. A S Purewal and R A Skurra> . 95 FEMS Microbiol Lett . pp 259-266 (1992) Their predicted transmembrane domains is four and these protems are not homologous to the large multidrug resistance proteins, or to other membrane transporters L Grmius et al , supra A typical example is a protein (Smr). encoded b} staphylococcal multidrug resistance gene, which contains about 107 ammo acid residues

The Smr gene is homologous to at least 25 different genes which encode three major pes of protein, including drug resistance proteins encoded b genes on transmissible plasmids located at the 3'-conserved segment of mtegrons and Smr itself L Gπnms. G Dreguniene. E B Goldberg D -H Liao and S J Pro_jan. 27 Plasmid. pp 119-129 (1992), I T Paulsen. T G Littlejohn P Radstrom, L Sundstrom, O Skold. G Swedberg and R A Skurray, 37 Antimicrob Agents Chemother . pp 761-768 (1993) and the chromosome-encoded E coh proteins responsible for resistance to aromatic dyes S Purewal 68 FEMS Microbiol Lett . pp 73-76 (1991). M Monmyo, E Hongo, H Hama-Inaja and I Machida 20 Nucleic Acids Res . pp 3159-3165 (1992)

The bacterial proteins responsible for the multidrug resistance are thought to be drug pumps which efflux drugs usmg an electrochemical proton gradient across the cytoplasmic membrane

However, these proteins are difficult to isolate and it would be advantageous to provide a simple method of isolation In addition, for the clinician, it would be advantageous to identify multidrug resistant bacteria before it is apparent that standard antibiotic therapy would be futile This invention answers both needs

OBJECTS OF THE INVENTION

It is an object of this invention to provide antibodies to the active drug resistant efflux pump protein

It is also an object of this invention to provide a method for rapid purification of drug resistance efflux pump proteins

It is also an object of this invention to provide an antibody, a method and a test kit using that antibody, for the clinician to provide rapid screening of cultures of potentially drug resistant pathogens

It is also an object of this invention to provide a method of immunization for such drug resistant pathogens

It is also an object of the present invention to provide a method for identifying compounds capable of binding to and/or inactivating the Smr protein

It is also an object of the present invention to provide a host cell which expresses the antibodies provided herein

It is also an object of the present invention to provide a method for identifying compounds active against multi-drug resistant pathogens

It is also an object of the present invention to provide an antibody useful in screening, in the isolation of the protein or as a targeting moiety for the protein

SUMMARY OF THE INVENTION

The invention relates to novel monoclonal antibodies to the Staphylococcal multidrug resistance (Smr) protein and a hybπdoma cell line which produces that antibody This invention detects strains of bacteria expressing Smr and related proteins The invention also aids in punfying the protein and in studying its mechanism of action, and may also provide the basis for a diagnostic to aid in identifying antibiotic-resistant bacteria

This invention provides a method for identifying compounds capable of binding to the Smr protein, and determining the amount and affinity of a compound capable of binding to the Smr protein in a sample

This invention also provides a host cell comprising a recombinant expression vector encoding the protein, fragment or mutant thereof, useful for these purposes

This invention also provides an in vivo or in vitro method for screening for drug resistant pathogens capable of manufacture and use in a kit form

Other aspects of this invention will be apparent as the description proceeds

DETAILED DESCRIPTION OF THE INVENTION

The following provides definitions for the terms used to described the present invention

The term "protein" refers to a Smr protein or a fragment thereof

The terms "antibody" and "anti-Smr antibody¹' each refer to any antibodies, or a fragment thereof, that have been produced using the Smr protein as an lmmugen These may be monoclonal or polyclonal antibodies (monoclonal are preferred), and can be from any of several sources The terms include recombinant. chimenc and affinity modifed forms made by techniques well known in the molecular biology art The invention also contemplates fragments of these antibodies made b} any method in the protein or peptide art

The term "clinical screen" refers to a screen for a multi-drug resistant pathogen Preferably this screen is used on infected body tissues or fluids of an animal or cell culture using standard techniques, such as ELISA It also contemplates "mapping" of disease in a whole body, such as b\ labeled antibody as described above given systemicalh Regardless of the detection method, preferably such detection methods include fluorescence. X-ray (including CAT scan), NMR (including MRI). and the like

The term "compound screen" refers to the methods and screens related to finding compounds, determining their affinity and inactivation of Smr protein for designing or selecting compounds based on the screen In another embodiment, it contemplates the use of the three dimensional structure for drug design, preferably "rational drug design", as understood by the art It may be preferred that the antibody is in "essentially pure form" which refers to an antibody reasonably free of other impurities, so as to make it useful for experiments or characterization Use of this screenmg method assists the skilled artisan in finding novel structures, whether made by the chemist or by nature, which bind to and preferably inhibit Smr protein These "inhibitors" may be useful in regulating or modulating the activity of the protein, and may be used to affect treatment provided by the use of antimicrobial agents This approach affords new pharmaceutically useful compounds

The skilled artisan will appreciate that the degeneracy of the genetic code provides for differing DNA sequences to provide the same transcnpts, and thus the same peptides, described herein In certain cases preparing the DNA sequence, which encodes for the same peptide, but differs from the native DNA include

• ease of sequencing or synthesis,

• increased expression of the peptide, and

• preference of certain heterologous hosts for certain codons over others

These practical considerations are widely known and provide embodiments that may be advantageous to the user of the invention Thus, it is clearly contemplated that the native DNA is not the only embodiment envisioned in this invention

In addition, it is apparent to the skilled artisan that fragments of the protein may be used in screening, drug design and the like, and that the entire peptide may not be required for the purposes of using the invention Thus it is clearly contemplated that the skilled artisan will understand that the disclosure of the peptide and its uses contemplates the useful peptide fragments of the peptide

The practical considerations of protein expression, purification yield, stability, solubility, and the like, are considered by the skilled artisan when choosing whether to use a fragment, and the fragment to be used As a result, using routine practices in the art, the artisan can, given this disclosure, practice the invention using fragments of the protein as well

The protein or protease itself can be used to determine the binding activity of small molecules to the protein Drug screening using enzymatic targets is used m the art and can be employed using automated, high throughput technologies

Antibodies used to identify a Smr protein and other members of this highly conserved protein family are purified and verified for activity using Western blotting and ELISA procedures Anti-Smr antibodies and fragments thereof (e g , Fabi and Fab₂) are used to crystallize Smr protein Smr's in active form can then be provided m quantities sufficient for immunization and reconstitution to be used in immunoassays, lmmunoblotting and immunoprecipitation The Smr antibody is unique in that segments of purified Smr do not interact with the antibody whereas the intact folded Smr protein does Hence the antibody has low or undetectable cross reactivity with bacterial proteins unrelated to Smr Without being bound by theory, it is thought that the antibody recognizes two or more nonsequential domains of the folded protein

Preparation and Use of Antibodies

Because Smr belongs to a Smr family of bacterial proteins, no Smr homologues have been identified in man or mouse Hence the Smr protein is amenable to antibody production by conventional methods

Antibodies may be made by several methods For example, the protein may be injected into suitable (e g . mammalian) subjects, including mice, rats, rabbits, and hamsters Preferred protocols involve repeated injection of the immunogen in the presence of adjuvants according to a schedule which boosts production of antibodies in the serum The titers of the immune serum can readily be measured using immunoassay procedures, now standard in the art

The antisera obtained can be used directly or monoclonal antibodies may be obtamed by harvesting the peripheral blood lymphocytes or the spleen of the immunized animal and immortalizing the antibody-producing cells, followed by identifying the suitable antibody producers usmg standard immunoassay techniques

Polyclonal or monoclonal preparations are useful in monitoring therapy or prophylaxis regimens involving the compounds of the invention Suitable samples such as those derived from blood, serum, urine, or saliva can be tested for the presence of the protein at various times during the treatment protocol using standard immunoassay techniques which employ the antibody preparations of the invention

These antibodies can also be coupled to labels such as scintigraphic labels, e g . technetium 99 or 1-131, using standard coupling methods However, a label can include use of any mateπal which directly or indirectly provides a means for detection The label may be joined to the antibody via covalent or non-covalent bonding The labeled compounds are administered to subjects to determine the locations of drug resistant bacterial infection in vivo Hence, a labeled antibody to the protein would operate as a screening tool for such diseases The ability of the antibodies to bind drug resistant bacteria selectively is thus taken advantage of to map the distπbution of these bacteria in situ The techmques can also be employed in histological procedures and the labeled antibodies can be used in competitive immunoassays

Antibodies are advantageously coupled to other compounds or mateπals usmg known methods For example, materials having a carboxyl functionality, the carboxyl residue can be reduced to an aldehyde and coupled to a carrier through reaction with side chain ammo groups, optionally followed by reduction of the lmino linkage formed The carboxyl residue can also be reacted with side chain ammo groups using condensing agents such as dicyclohexyl carbodiimide or other carbodiimide dehydrating agents Linker compounds can also be used to effect the coupling, both homobifunctional and heterobifunctional linkers are available from Pierce Chemical Company, Rockford, 111

These antibodies, when conjugated to a suitable chromatography mateπal, are useful in isolating the Smr protein Separation methods using affinity chromatography are well known in the art, and are within the purview of the skilled artisan

Disease marker

In addition, when used in a disease screen, antibodies can be conjugated to chromophore- or fluorophore-containing materials, or can be conjugated to enzymes which produce chromophores or fluorophores in certain conditions These conjugating materials and methods are well known in the art When used in this manner, detection of the protein by immunoassay is straightforward to the skilled artisan Body fluids, for example can be screened m this manner for calibration, and detection of distribution of drug resistant bacteria, or increased levels of these bacteria

When used in this way the invention is a useful diagnostic and or clinical marker for drug resistant bacterial mfection When disease is detected, it may be treated before the onset of symptom or debilitation

Furthermore, such antibodies can be used to target diseased tissue, for detection or treatment as described above

EXAMPLES

The following non-limiting examples illustrate a prefeπed embodiment of the present invention, and bπefly describe the uses of the present invention These examples are provided for the guidance of the skilled artisan, and do not limit the invention in any way Armed with this disclosure and these examples the skilled artisan is capable of making and using the claimed invention

Standard starting materials are used for these examples Many of these materials are known and are commercially available For example, known plasmids and Kunkel method mutagenesis is well known in the art

Variants may be made by expression systems and by various methods in various hosts, these methods are withm the scope of the practice of the skilled artisan in molecular biology, biochemistry or other arts related to biotechnology

Example 1 A The FLAG epitope is attached to the C-terminus of the Smr protein The Smr protein is overexpressed in E coh and purified on the anti-FLAG affinity column Smr protein is purified in a volume of 1 2 ml The protein is dissolved in a buffer containing 0 1 M Glycme (pH 6-7), 1 2% octylglucoside, and 8% glycerol

B Mice are injected with desired Smr protein (500-200 ug) and complete Freund's adjuvant in a 1 1 mixture Five mice are immunized mtrapeπtoneally (IP) or subcutaneously (SC) with 300-500 μl of mixture

After 2-3 weeks, mice are injected IP with incomplete Freund's adjuvant Seven to ten days after the 2nd immunization, mouse serum is collected and analyzed on a dot blot or ELISA Preimmune serum and a positive control are also tested Mice receive a third injection IP at 4-6 weeks Mouse serum is collected 7-10 days post-third-injection and checked for lmmunoreactivity to antigen by ELISA. dot blot oi immunoprecipitation One mouse displays a good response to the antigen, and as the best responder. it is boosted at 8-10 weeks, via IV and/or IP routes with saline and incomplete Freund's adjuvant respectively Three days after the final injection (approximately 60-75 days from the initial injection), the fusion is performed

Screening for positive hybπdomas begins 7-10 days after the fusion and continues up to 21 days Selected positives are expanded and stored in liquid nitrogen In addition, limiting dilutions are performed for single cell cloning Hence, from the initial immunization to a cloned hybπdoma line takes approximately a total of 110-130 days Selected hybπdomas are cultured for large scale puπfication or are injected mto mice to generate ascites fluid Purity of antibody is determined based on its use

C Hybπdomas producing anti-Smr antibodies are identified using ELISA with antibodies of interest are run on two sets of 96-well plates one covered with the Smr-FLAG and another covered with the NorA-FLAG protein Only antibodies which interact with the Smr-FLAG, but do not interact with the NorA-FLAG or Bovine alkaline phosphatase- FLAG, BAP-FLAG, are used for further analysis

D Tissue culture supernatant is affinity purified against antigen affixed to a column material

(In addition, an Anti-IgG, Protein A or G column provides acceptable purification ) To purify anti-Smr antibodies further, the Smr-FLAG protein is absorbed on the anti-FLAG affinity gel Anti-Smr antibodies are dissociated from the column at pH 4 and neutralized with Tπs pH 8 0 The Smr-FLAG (15kDa) is removed from the antibody preparation by ultrafiltration

Example 2 The hybπdoma cell line (antibody-producing cells) is generated by fusing mouse spleen cells and mouse P3X-X63Ag8 myeloma cells The mouse spleen cells are isolated from a Balb/c mouse immunized with an Smr fusion protein to which is attached a non-removable 9 amino acid FLAG peptide Hybπdomas are selected using a hypoxanthine/aminopteπn/thymidine medium These hybπdomas are selected for their ability to produce antibody which recognizes Smr protein coated on 96 wells and on PVDF membrane blotted with Smr-FLAG protein, and which does not recognize an unrelated protein tagged with FLAG

Example 3 An isolated monoclonal antibody (clone P4B7G6) is made against, Smr protein which is produced as a fusion protein (plasmid pSE380smrFLAG) The Smr protein is from the E coh KOI 489 bacteria that has not been described m the literature The hydrophobic Smr (membrane protein) immunogen stimulates an immune reaction in mice The resulting antibody has the ability to quickly identify resistant strains of bacteria and assist in the course of treatment

Example 4 The antibody is used in ELISA to determine the prevalence and spread of Smr-related resistance

Example 5 The monoclonal antibody of Example 1 is used in column chromatography allowing rapid isolation of Smr and Smr-related proteins to perform mechanistic studies

Example 6 The antibody of Example 1 is used to evaluate the bacterial samples m clinical samples from patients suspected of mfection from strains of bacteria resistant to available drugs targetmg the efflux pump composed of Smr-related proteins The sample is tested using standard ELISA procedures

While it is believed that the disclosure hereinabove is sufficient to teach the preparation of hybridoma cell line of the present invention (P4B7G6), the cell line was also deposited with the American Type Culture Collection and is designated as Patent Deposit PTA-423 All references described herein are hereby incorporated by reference While particular embodiments of the subject invention have been described, it will be obvious to those skilled in the art that various changes and modifications of the subject invention can be made without departing from the spirit and scope of the invention It is intended to cover, in the appended claims, all such modifications that are within the scope of this invention

Claims

WHAT IS CLAIMED IS:

1. An antibody to the Staphylococcal multidrug resistance (Smr) protein, or a fragment of the antibody.

2. The antibody of Claim 1, characterized in that the antibody is the antibody produced by the hybridoma cell line, P4B7G6, which is deposited with the American Type Culture Collection as Patent Deposit PTA-423.

3. The antibody according to any one of the preceding claims, characterized in that the antibody is in essentially pure form.

4. The antibody according to Claim 1 or 3, characterized in that the antibody is monoclonal.

5. A DNA sequence which encodes the antibody of any one of the preceding claims.

6. The antibody according to any one of Claims 1, 2, 3 or 4, characterized in that the antibody has been labeled with a detectable label.

7. The use of the antibody according to any one of Claims 1, 2, 3, 4 or 6, for screening for drug resistant pathogens in a sample.

8. The use according to Claim 7 characterized in that the sample is blood, saliva, synovial fluid or other body fluids.

9. A screening kit for identifying drug resistant pathogens, comprising the antibody according to of any one of Claim 1, 2, 3, 4 or 6.

10. A hybridoma cell line that produces the antibody of Claim 1 or 2.